Novel LHRH antagonists having improved solubility properties

ABSTRACT

The invention relates to peptides which contain N-methylated amino acid units and have improved water solubility. Medicaments in which the peptides according to the invention are contained can be used for the treatment of hormone-dependent tumours and hormone-influenced non-malignant disorders.

[0001] The invention relates to LHRH antagonists having improvedsolubility properties, processes for the preparation of these compounds,medicaments in which these compounds are contained, and the use of themedicaments for the treatment of hormone-dependent tumours andhormone-influenced non-malignant disorders such as benign prostatehyperplasia (BPH) and endometriosis.

[0002] The nomenclature used for the definition of the peptides agreeswith that nomenclature explained by the IUPAC-IUB Commission onBiochemical Nomenclature (European J. Biochem. 1984, 138, 9-37), inwhich, in agreement with the conventional representation, the aminogroups at the N terminus appear to the left and the carboxyl group atthe C terminus appears to the right. The LH-RH antagonists such as thepeptides according to the invention include naturally occurring andsynthetic amino acids, the former including Ala, Val, Leu, Ile, Ser,Thr, Lys, Arg, Asp, Asn, Glu, Gln, Cys, Met, Phe, Tyr, Pro, Trp and His.The abbreviations for the individual amino acid residues are based onthe trivial names of the amino acids and are Ala=alanine, Arg=arginine,Gly=glycine, Leu=leucine, Lys=lysine, Pal(3)=3-(3-pyridyl)alanine,Nal(2)=3-(2-naphthyl)-alanine, Phe=phenylalanine,Cpa=4-chlorophenylalanine, Pro=proline, Ser=serine, Thr=threonine,Trp=tryptophan, Try=tyrosine and Sar=sarcosine. All amino acidsdescribed here originate from the L series, if not mentioned otherwise.For example, D-Nal(2) is the abbreviation for 3-(2-naphthyl)-D-alanineand Ser is the abbreviation for L-serine. Substitutions on the ε aminogroup in the side chain of lysine are represented by a term placed inbrackets behind Lys, if appropriate in the form of an abbreviation.

[0003] Other abbreviations used are:

[0004] Ac Acetyl

[0005] Atz 3-Amino-1,2,4-triazole-5-carbonyl

[0006] B 4-(4-Amidinophenyl)amino-1,4-dioxobutyl

[0007] Boc tert-Butyloxycarbonyl

[0008] Bop Benzotriazol-1-oxy-tris(dimethylamino)-phosphoniumhexafluorophosphate

[0009] DCC Dicyclohexylcarbodiimide

[0010] DCM Dichloromethane

[0011] Ddz Dimethoxyphenyl-dimethylmethylenoxy-carbonyl(Dimethoxy-dimethyl-Z)

[0012] DIC Diisopropylcarbodiimide

[0013] DIPEA N,N-Diisopropylethylamine

[0014] DMF Dimethylformamide

[0015] Fmoc Fluorenylmethyloxycarbonyl

[0016] HF Hydrofluoric acid

[0017] HOBt 1-Hydroxybenzotriazole

[0018] HPLC High-pressure liquid chromatography

[0019] Me Methyl

[0020] TFA Trifluoroacetic acid

[0021] Z Benzyloxycarbonyl

[0022] The peptides according to the invention are analogues of theluteinizing-hormone-releasing hormone (LH-RH), which has the followingstructure: p-Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH₂, [LH-RH,gonadorelin].

[0023] For more than 20 years, researchers have sought selective potentantagonists of the LH-RH decapeptide [M. Karten and J. E. Rivier,Endocrine Reviews 7, 44-66 (1986)]. The high interest in suchantagonists is based on their usefulness in the field of endocrinology,gynaecology, contraception and cancer. A large number of compounds havebeen prepared as potential LH-RH antagonists. The most interestingcompounds which have been found to date are those compounds whosestructures are a modification of the LH-RH structure.

[0024] The first series of potent antagonists was obtained by theintroduction of aromatic amino acid residues into the positions 1, 2, 3and 6 or 2, 3 and 6. The customary way of writing the compounds is asfollows: the amino acids are first indicated which have taken the placeof the amino acids originally present in the peptide chain of LH-RH, thepositions in which the exchange took place being marked by superscriptedfigures. Furthermore, by the notation “LH-RH” placed afterwards it isexpressed that these are LH-RH analogues in which the exchange has takenplace.

[0025] Known antagonists are:

[0026] [Ac-D-Cpa^(1,2), D-Trp^(3,6)] LH-RH (D. H. Coy et al., In: Gross,E. and Meienhofer, J. (Eds) Peptides; Proceedings of the 6th AmericanPeptide Symposium, pp. 775-779, Pierce Chem. Co., Rockville III. (1979):[Ac-Pro¹, D-Cpa², D-Nal(2)^(3,6)] LH-RH (U.S. Pat. No. 4,419,347) and[Ac-Pro¹, D-Cpa², D-Trp^(3,6)] LH-RH (J. L. Pineda, et al., J. Clin.Endocrinol. Metab. 56, 420, 1983).

[0027] In order to improve the action of antagonists, basic amino acids,for example D-Arg, were later introduced into the 6 position. Forexample [Ac-D-Cpa^(1.2), D-Trp³, D-Arg⁶, D-Ala¹⁰] LH-RH(ORG-30276) (D.H. Coy, et al., Endocrinology 100, 1445, 1982); and

[0028] [Ac-D-Nal(2)1, D-Phe(4-F)², D-Trp³, D-Arg⁶] LH-RH(ORF 18260) (J.E. Rivier et al., in: Vickery B. H. Nestor, Jr. J. J., Hafez, E. S. E(Eds). LHRH and its Analogs, pp. 11-22 MTP Press, Lancaster, UK 1984).

[0029] Further potent LH-RH antagonists are described in WO 92/19651, WO94/19370, WO 92/17025, WO 94/14841, WO 94/13313, U.S. Pat. No.5,300,492, U.S. Pat. No. 5,140,009, EP 0 413 209 A1 and DE 195 44 212A1.

[0030] The latter discloses compounds having a modified ornithine orlysine unit in position 6 and which correspond to the following formula:

Ac-D-Nal(2)¹-D-Cpa²-D-Pal(3)³-Ser⁴-Tyr⁵-D-Xxx⁶-Leu⁷-Arg⁸-Pro⁹-D-Ala¹⁰-NH₂,

[0031] in which D-Xxx is an amino acid group of the general formula (VI)

[0032] Further known LH-RH antagonists are antarelix, ganirelix andcetrorelix.

[0033] Antarelix:

[0034] Ac-D-Nal(2)¹-D-Cpa²-D-Pal(3)³-Ser⁴-Tyr⁵-D-Hci⁶-Leu⁷-Lys(iPr)⁸-Pro⁹-D-Ala¹⁰-NH₂

[0035] Ganirelix:

[0036] Ac-D-Nal(2)¹-D-Cpa²-D-Pal(3)³-Ser⁴-Tyr⁵-D-hArg(Et)₂⁶-Leu⁷-hArg(Et)₂ ⁸-Pro⁹-D-Ala¹⁰-NH₂

[0037] Cetrorelix:

[0038]Ac-D-Nal(2)¹-D-Cpa²-D-Pal(3)³-Ser⁴-Tyr⁵-D-Cit⁶-Leu⁷-Arg⁸-Pro⁹-D-Ala¹⁰-NH₂.

[0039] The aim of the invention is to create novel LH-RH antagonistswhich have an increased enzymatic stability and significantly improvedwater solubility.

[0040] This object is achieved by compounds of the following generalformula (I)

[0041] A-Xxx¹-Xxx²-Xxx³-Xxx⁴-Xxx⁵-Xxx⁶-Xxx⁷-Xxx⁸-Xxx⁹-Xxx¹⁰-NH₂  (I)

[0042] in which

[0043] A is an acetyl or a 3-(4-fluorophenyl)propionyl group,

[0044] Xxx¹ is D-Nal(1) or D-Nal(2),

[0045] Xxx²-Xxx³ is D-Cpa-D-Pal(3) or a single bond,

[0046] Xxx⁴ is Ser,

[0047] Xxx⁵ is N-Me-Tyr,

[0048] Xxx⁶ is D-Cit, D-Hci or a D-amino acid group of the generalformula (II)

[0049] in which n is the number 3 or 4, where R¹ is a group having thegeneral formula III

—(CH ₂)_(p)—CO—NR²R³  (III)

[0050] where p is an integer from 1 to 4, R² is hydrogen or an alkylgroup and R³ is an unsubstituted or substituted aryl group or heteroarylgroup, or R¹ is a 3-amino-1,2,4-triazole-5-carbonyl group or R¹ is aring of the general formula (IV)

[0051] in which q is the number 1 or 2, R⁴ is a hydrogen atom or analkyl group, R⁵ is a hydrogen atom or an alkyl group and X is an oxygenor sulphur atom,

[0052] Xxx⁷ is Leu or Nle,

[0053] Xxx⁸ is Arg or Lys(iPr),

[0054] Xxx⁹ is Pro and

[0055] Xxx¹⁰ is Ala, D-Ala or Sar,

[0056] and their salts with pharmaceutically acceptable acids, inparticular the acetates, embonates and trifluoroacetates.

[0057] Among the compounds according to the invention, those areparticularly preferred in which Xxx⁶ isD-[ε-N′-(imidazolidin-2-on-4-yl)formyl]-Lys,D-(3-amino-1,2,4-triazole-3-carbonyl)-Lys, abbreviated D-Lys(Atz) orD-[ε-N′-4-(4-Amidinophenyl)-amino-1,4-dioxo-butyl]-Lys, abbreviatedD-Lys(B).

[0058] Further particularly preferred compounds according to theinvention are:

[0059]Ac-D-Nal(2)¹-D-Cpa²-D-Pal(3)³-Ser⁴-N-Me-Tyr⁵-D-Hci⁶-Nle⁷-Arg⁸-Pro⁹-D-Ala¹⁰-NH₂,

[0060] Ac-D-Nal(2)¹-D-Cpa²-D-Pal(3)³-Ser⁴-N-Me-Tyr⁵-D-Lys(Atz)⁶-Leu⁷-Arg⁸-Pro⁹-D-Ala¹⁰-NH₂,

[0061] Ac-D-Nal(2) 1-D-Cpa²-D-Pal(3)³-Ser⁴-N-Me-Tyr⁵-D-Lys(B)-Leu⁷-Lys(iPr)⁸-Pro⁹-D-Ala¹⁰-NH₂,

[0062] Ac-D-Nal(2)²-D-Cpa²-D-Pal(3)3-Ser⁴-N-Me-Tyr⁵-D-Lys(B)⁶-Leu⁷-Arg⁸-Pro⁹-D-Ala¹⁰-NH₂,

[0063]Ac-D-Nal(2)¹-D-Cpa²-D-Pal(3)³-Ser⁴-N-Me-Tyr⁵-D-Hci⁶-Nle⁷-Lys(iPr)⁸-Pro⁹-D-Ala¹⁰-NH₂,

[0064]Ac-D-Nal(2)¹-D-Cpa²-D-Pal(3)³-Ser⁴-N-Me-Tyr⁵-D-Hci⁶-Nle⁷-Lys(iPr)⁸-Pro⁹-Sar¹⁰-NH₂,

[0065] Ac-D-Nal(2)¹-D-Cpa²-D-Pal(3)³-Ser⁴-N-Me-Tyr⁵-D-Hci⁶-Nle⁷-Arg⁸-Pro⁹-Sar¹⁰-NH₂,

[0066] 3-(4-Fluorophenyl)propionyl-D-Nal(1)¹-Ser⁴-N-Me-Tyr⁵-D-Lys(Atz)⁶-Leu⁷-Arg⁸-Pro⁹-D-Ala¹⁰-NH₂,

[0067]Ac-D-Nal(2)¹-D-Cpa²-D-Pal(3)³-Ser⁴-N-Me-Tyr⁵-D-Lys(B)⁶-Nle⁷-Arg⁸-Pro⁹-Sar¹⁰-NH₂,

[0068]Ac-D-Nal(2)¹-D-Cpa²-D-Pal(3)³-Ser⁴-N-Me-Tyr⁵-D-Lys(B)⁶-Nle⁷-Arg⁸-Pro⁹-D-Ala¹⁰-NH₂and

[0069] Ac-D-Nal(2)¹-D-Cpa²-D-Pal(3)³-Ser⁴-N-Me-Tyr⁵-D-Lys(B)⁶-Nle⁷-Lys(iPr)⁸-Pro⁹-Sar¹⁰-NH₂

[0070] and also their salts with the above-mentioned pharmaceuticallyacceptable acids.

[0071] The compounds according to the invention can be used for thetreatment of hormone-dependent tumours, in particular prostate carcinomaor breast cancer, and also for non-malignant indications whose treatmentnecessitates LH-RH hormone suppression. For this, they are mixed withthe customary vehicles and excipients and formulated as medicaments.

[0072] The synthesis of compounds according to formula (I) can both becarried out either by classical fragment condensation or by solid-phasesynthesis according to Merrifield with synthesis following one anotherusing D-lysine already acylated in the side chain with the carboxylicacid of the general formula R¹-COOH or by reaction of a decapeptide unitwith the appropriate carboxylic acids by amide linkage in the side chainof D-lysine 6. Accordingly, the introduction of the R¹—CO-group can beperformed in three different positions in the process: before thecondensation of the individual units to give the peptide, after theincorporation of lysine or ornithine in the peptide chain, but beforethe condensation of the next unit or after condensation of all units.

[0073] The compounds of the formula (I) are synthesized according to theknown methods, such as, for example, by pure solid-phase technique,partly solid-phase technique (so-called fragment condensation) or by theclassical solution couplings (see M. Bodanszky, “Principles of PeptideSynthesis”, Springer Verlag 1984).

[0074] For example, the methods of solid-phase synthesis are describedin the textbook “Solid Phase Peptide Synthesis”, J. M. Stewart and J. D.Young, Pierce Chem. Company, Rockford, III, 1984, and in G. Barany andR. B. Merrifield “The Peptides”, Ch. 1, pp. 1-285, 1979, Academic PressInc. Classical solution syntheses are described in detail in thetreatment “Methoden der Organischen Chemie [Methods of OrganicChemistry] (Houben-Weyl), Synthese von Peptiden” [Synthesis of Peptides]E. Wunsch (Editor) 1974, Georg Thieme Verlag, Stuttgart, FRG.

[0075] The stepwise synthesis is carried out, for example, by firstcovalently bonding the carboxy-terminal amino acid whose α-amino groupis protected to an insoluble support which is customary for this,removing the α-amino protective group of this amino acid, bonding thefree amino group thus obtained to the next protected amino acid via itscarboxyl group, and in this manner linking the customary amino acids ofthe peptide to be synthesized in the correct sequence step for step, andafter linkage of all amino acids removing the finished peptide from thesupport and removing any further side function protective groups whichmay be present. The stepwise condensation is carried out in aconventional manner by synthesis from the corresponding, customarilyprotected amino acids.

[0076] The linkage of the individual amino acids to one another iscarried out according to the methods customary for this; thoseparticularly suitable are:

[0077] Symmetrical anhydride method in the presence ofdicyclohexylcarbodiimide or diisopropylcarbodiimide (DCC, DIC)

[0078] Carbodiimide method generally

[0079] Carbodiimide/hydroxybenzotriazole method

[0080] (see The Peptides, Volume 2, Ed. E. Gross and J. Meienhofer)

[0081] In the fragment coupling, the azide coupling, which proceedswithout racemization, or the DCC-1-hydroxybenzotriazole orDCC-3-hydroxy-4-oxo-3,4-dihyro-1,2,3-benzotriazine method is preferablyused. Activated esters of fragments can also be employed.

[0082] Esters of N-protected amino acids, such as, for example,N-hydroxysuccinimide esters or 2,4,5-trichlorophenyl esters, areparticularly highly suitable for the stepwise condensation of aminoacids. The aminolysis can be very well catalysed by N-hydroxy compoundswhich have approximately the acidity of acetic acid, such as, forexample, 1-hydroxybenzotriazole.

[0083] Intermediate amino protective groups which present themselves aregroups which are removed by hydrogenation, such as, for example, thebenzyloxycarbonyl radical (=Z radical) or groups which can be removed byweak acid. Suitable protective groups for the α-amino groups are, forexample: tertiary butyloxycarbonyl groups, fluorenylmethyloxycarbonylgroups, carbobenzoxy groups or carbobenzothio groups (if appropriate ineach case having a p-bromo- or p-nitrobenzyl radical), thetrifluoroacetyl group, the phthalyl radical, the o-nitrophenoxyacetylgroup, the trityl group, the p-toluenesulphonyl group, the benzyl group,benzyl radicals substituted in the benzene nucleus (p-bromo- orp-nitrobenzyl radical) and the α-phenylethyl radical. Reference is alsomade here to P. Greenstein and Milton Winitz, Chemistry of Amino Acids,New York 1961, John Wiley and Sons, Inc., Volume 2, for example page 883et seq., “Principles of Ppetide Synthesis”, Springer Verlag 1984, “SolidPhase Peptide Synthesis”, J. M. Stewart and J. D. Young, Pierce Chem.Company, Rockford, III, 1984, G. Barany and R. B. Merrifield “ThePeptides”, Ch. 1, pp. 1-285, 1979, Academic Press Inc., and also ThePeptides, Volume 2, Ed. E. Gross and J. Maienhofer, Academic Press, NewYork. These protective groups are fundamentally also suitable for theprotection of further functional side groups (OH groups, NH₂ groups) ofthe corresponding amino acids.

[0084] Hydroxyl groups present (serine, threonine) are preferablyprotected by benzyl groups and similar groups. Further amino groups notin the α-position (for example amino groups in the ω-position, guanidinogroup of arginine) are preferably orthogonally protected.

[0085] The individual amino acid units, excluding lysine or ornithinemodified by the R¹—CO-group, are commercially obtainable. A possiblecourse of the process for the preparation of the latter compounds is asfollows:

[0086] 1. The α-carboxylic acid group is amidated.

[0087] 2. The ε-amino group is protected by the Z group.

[0088] 3. The α-amino group is protected by the Boc group such that aselectivity with respect to the later removal of the amino protectivegroups results.

[0089] 4. The Z group on the ε-amino group is removed.

[0090] 5. The desired group R⁴—Co— is introduced on the ε-amino group.

[0091] 6. The Boc group on the α-amino group is removed.

[0092] 7. The α-amino group is provided with the Z group.

[0093] For the introduction of the R¹—CO-group by reaction of the aminogroup of the lysine with appropriate carboxylic acid, suitable processesare fundamentally the same processes as described above for the linkageof the amino acids. However, condensation using carbodiimide, forexample 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, and1-hydroxybenzotriazole is particularly preferred.

[0094] The reaction for the linkage of the amino acids takes place in aninert solvent or suspending agent which is customary for this (forexample dichloromethane), it being possible to add dimethylformamide, ifnecessary, to improve the solubility.

[0095] Suitable synthetic supports are insoluble polymers, for examplepolystyrene resin in bead form, which can be swollen in organic solvents(for example a copolymer of polystyrene and 1% divinylbenzene). Thesynthesis of a protected decapeptide amide on a methylbenzhydrylamineresin (MBHA resin, i.e. polystyrene resin provided withmethylbenzhydrylamine groups), which affords the desired C-terminalamide function of the peptide after HF cleavage from the support, can becarried out according to the following flow diagram:

[0096] Flow Diagram

[0097] Peptide Synthesis Protocol Stage Function Solvent/Reagent (v/v)Time 1 Washing Methanol 2 × 2 min 2 Washing DCM 3 × 3 min 3 RemovalDCM/TFA (1:1) 1 × 30 min  4 Washing Isopropanol 2 × 2 min 5 WashingMethanol 2 × 2 min 6 Washing DCM 2 × 3 min 7 Neutralization DCM/DIPEA(9:1) 3 × 5 min 8 Washing Methanol 2 × 2 min 9 Washing DCM 3 × 3 min 10STOP Addition of the Boc-As in DCM + DIC + HOBt 11 Coupling DCM,optionally DCM/DCF approx. 90 min 12 Washing Methanol 3 × 2 min 13Washing DCM 2 × 3 min

[0098] The Nα-Boc-protected amino acids are customarily coupled in athree fold molar excess in the presence of diisopropylcarbodiimide (DIC)and 1-hydroxybenzotriazole (HOBt) in CH₂Cl₂/DMF in the course of 90 min,and the Boc-protected group is removed by action of 50% trifluoroaceticacid (TFA) in CH₂Cl₂ for half an hour. To check for complete conversion,the chloranil test according to Christensen and the Kaiser's ninhydrintest can be used. Radicals of free amino functions are blocked byacetylation in a five fold excess of acetylimidazole in CH₂Cl₂. Thesequence of the reaction steps of the peptide synthesis on the resinfollows from the flow diagram. For the removal of the resin-boundpeptides, the respective final product of the solid phase synthesis isdried in vacuo over P₂O₅ and treated at 0° C. for 60 min in a 500-foldexcess of HF/anisole 10:1/v:v.

[0099] After distilling of HF and anisole in vacuo, the peptide amidesare obtained as white solids by washing with anhydrous ethyl ether withstirring, and the removal of polymeric support additionally obtained iscarried out by washing with 50% strength aqueous acetic acid. By carefulconcentration of the acetic acid solutions in vacuo, the respectivepeptides can be obtained as highly viscous oils, which are convertedinto white solids after addition of abs. ether in the cold.

[0100] Further purification is carried out by routine methods ofpreparative high-pressure liquid chromatography (HPLC).

[0101] The conversion of the peptides into their acid addition salts canbe effected in a manner known per se by reaction thereof with acids.Conversely, free peptides can be obtained by reaction of their acidaddition salts with bases. Peptide embonates can be prepared by reactionof trifluoroacetic acid salts (TFA salts) of the peptide with freeembonic acid (pamoic acid) or the corresponding disodium salt of embonicacid. For this, the peptide TFA salt is treated in aqueous solution withthe solution of disodium embonate in polar aprotic medium, preferablydimethylacetamide, and the pale yellow precipitate formed is isolated.

[0102] The following examples serve to illustrate the invention withoutrestricting it.

EXAMPLE 1

[0103]Ac-D-Nal(2)¹-D-Cpa²-D-Pal(3)³-Ser⁴-N-Me-Tyr⁵-D-Hci⁶-Nle⁷-Arg⁸-Pro⁹-D-Ala¹⁰-NH₂

[0104] The synthesis was carried out according to a solid-phase flowdiagram (Peptide Synthesis Protocol, p. 11) with DIC/HOBt coupling,starting from 3.3 g of MBHA resin (loading density 1.08 mmol/g). AfterHF cleavage from the polymeric support, 3.4 g of crude peptide wereobtained, which were purified by standard processes of preparative HPLC.After subsequent freeze-drying, 1.43 g of HPLC-uniform product of theempirical formula C₇₂H₉₆N₁₇O₁₄Cl having correct FAB-MS: 1458.7 (M+H⁺)(calc: 1457.7), and corresponding ¹H-NMR spectrum were obtained.

[0105]¹H-NMR (500 MHz, D₂O/DMSO-d₆, δ in ppm) 8.7 to 7.2, several m,arom. H and incompletely exchanged NH; 6.92 and 6.58, 2d, 2×2H, arom. Hp-Cl-Phe; 5.2 to 3.5, several m, Cα-H and aliph. H; 3.2 to 2.6, severalm, aromat. Cβ—H 2.1 to 0.7, several m, residual aliphat. H; 1.70, s, 3H,acetyl; 1.20, d, 3H, Cβ-H Ala; 0.8, m, Cδ-H Leu

EXAMPLE 2

[0106]Ac-D-Nal(2)¹-D-Cpa²-D-Pal(3)³-Ser⁴-N-Me-Tyr⁵-D-Lys(B)⁶-Leu⁷-Lys(iPr)⁸-Pro⁹-D-Ala¹⁰-NH₂

[0107] The synthesis was carried out according to a flow diagram(Peptide Synthesis Protocol, p. 11) with DIC/HOBt coupling, startingfrom 4.0 g of MBHA resin (loading density 1.11 mmol/g). After HFcleavage from the polymeric support, 4.87 g of crude peptide wereobtained, which were purified by standard processes of preparative HPLC.After subsequent freeze-drying, 0.93 g of HPLC-uniform product wasobtained, which was reacted with 4-amidinophenylamino-4-oxobutyric acidin the presence of BOP as a coupling reagent to give the desiredcompound. After fresh HPLC purification, 148 mg of target compound ofthe empirical formula C₈₅H₁₁₂N₁₇O₁₅—Cl having correct ESI-MS: 1647.6(M+H⁺) (calc: 1645.8), and corresponding ¹H-NMR spectrum were obtained.

[0108]¹H-NMR (500 MHz, DMSO-d₆, δ in ppm)

[0109] 10.4, s, 1H and 9.13, s, 2H, and 8.94, s, 2H, NHs of4-amidinoaniline; 8.6 to 7.35, several m, arom. H and NH; 7.22 and 7.18,2d, 4H, arom. H (pCl)Phe; 6.95 and 6.58, 2d, 4H, arom. H Tyr; 5.2 to3.5, several m, Cα-H and aliphat. H; 3.3 to 2.4, several m, Cβ-H andN—CH₃; 2.1 to 1.1, several m, residual aliphat. H; 1.68, s, 3H, acetyl;1.20, d, 3H, Cβ-H Ala; 0.83, dd, 6H, Cδ-H Leu

EXAMPLE 3

[0110] Ac-D-Nal(2)¹-D-Cpa²-D-Pal(3)³-Ser⁴-N-Me-Tyr⁵-D-Lys(B)-Leu⁷-Arg⁸-Pro⁹-D-Ala¹⁰-NH₂

[0111] The synthesis was carried out according to a solid-phase flowdiagram (Peptide Synthesis Protocol, p. 11) with DIC/HOBt coupling,starting from 4.0 g of MBHA resin (loading density 0.97 mmol/g). AfterHF cleavage from the polymeric support, 4.0 g of crude peptide wereobtained, which were purified by standard processes of preparative HPLC.After subsequent freeze-drying, 1.39 g of HPLC-uniform product wereobtained, which were reacted with 4-amidinophenylamino-4-oxobutyric acidin the presence of BOP as a coupling reagent to give the desiredcompound. After fresh HPLC purification, 440 mg of target compound ofthe empirical formula C₈₂H₁₀₆N₁₉O₁₅Cl having correct ESI-MS: 1632.7(M+H⁺) (calc: 1631.7), and corresponding ¹H-NMR spectrum were obtained.

[0112]¹H-NMR (500 MHz, DMSO-d₆, δin ppm):

[0113] 10.4, s, 1H and 9.15, s, 2H, and 9.0, s, 2H, NHs of4-amidinoaniline; 8.60, m, 2H, arom. H; 8.3 to 7.2, several m, arom. Hand NH; 7.27 and 7.20, 2d, 4H, arom. H (pCl)Phe; 6.96 and 6.60, 2d, 4H,arom. H Tyr; 5.2 to 3.5, several m, Cα-H and aliphat. H; 3.2 to 2.4,several m, Cβ-H and N—CH₃; 2.1 to 1.1, several m, residual aliphat. H;1.70, s, 3H, acetyl; 1.20, d, 3H, Cβ-H Ala; 0.85, dd, 6H, Cδ-H Leu

EXAMPLE 4

[0114]Ac-D-Nal(2)¹-D-Cpa²-D-Pal(3)³-Ser⁴-N-Me-Tyr⁵-D-Hci⁶-Nle⁷-Lys(iPr)⁸-Pro⁹-D-Ala¹⁰-NH₂

[0115] The synthesis was carried out according to a solid-phase flowdiagram (Peptide Synthesis Protocol, p. 11) with DIC/HOBt coupling,starting from 2.5 g of MBHA resin (loading density 1.08 mmol/g). AfterHF cleavage from the polymeric support, 2.78 g of crude peptide wereobtained, which were purified by standard processes of preparative HPLC.After subsequent freeze-drying, 400 mg of HPLC-uniform product of theempirical formula C₇₅H₁₀₂N₁₅O₁₄Cl having correct ESI-MS: 1472.6 (M+H⁺)(calc: 1471.7), and corresponding ¹H-NMR spectrum were obtained.

[0116]¹H-NMR (500 MHz, D₂O/DMSO-d₆, δ in ppm):

[0117] 8.62, m, 2H, 8.30, m, 2H, 7.80, m, 4H, 7.66, s, 1H, 7.47, m, 2H,7.36, d, 1H, aromat. H; 7.25 and 7.20, 2 d, 4H, arom. H (pCl) Phe; 6.96and 6.63, 2d, 4H, aromat. H Tyr; 5.10 to 4.0, several m, Cα-H andaliphat. H; 3.75 to 2.65, several m, Cβ-H and N—CH₃; 2.1 to 1.05,several m, residual aliphat. H; 1.74, s, 3H, acetyl; 1.23, d, 3H, Cβ-HAla; 1.20, m, CH₃ isoprop.; 0.8, m, 3H, Cδ-H Nle

EXAMPLE 5

[0118]Ac-D-Nal(2)¹-D-Cpa²-D-Pal(3)³-Ser⁴-N-Me-Tyr⁵-D-Hci⁶-Nle⁷-Lys(iPr)⁸-Pro⁹-Sar¹⁰-NH₂

[0119] The synthesis was carried out according to a solid-phase flowdiagram (Peptide Synthesis Protocol, p. 11) with DIC/HOBt coupling,starting from 2.5 g of MBRA resin (loading density 1.08 mmol/g). AfterHF cleavage from the polymeric support, 2.74 g of crude peptide wereobtained, which were purified by standard processes of preparative HPLC.After subsequent freeze-drying, 840 mg of HPLC-uniform product of theempirical formula C₇₅H₁₀₂N₁₅O₁₄Cl having correct ESI-MS: 1472.6 (M+H⁺)(calc: 1471.7), and corresponding ¹H-NMR spectrum were obtained.

[0120]¹H-NMR (500 MHz, D₂O/DMSO-d₆, δ in ppm):

[0121] 8.6, m, 2H, 8.3, m, 2H, 7.85, m, 2H, 7.8, m, 2H, 7.65, s, 1H,7.46, m, 2H, 7.35, d, 1H, aromat. H; 7.23 and 7.17, 2 d, 4H, arom. H(pCl)Phe; 7.0 and 6.6, 2d, 4H, aromat. H Tyr; 5.10 to 3.8, several m,Cα-H and aliphat. H; 3.75 to 2.6, several m, Cβ-H and N—CH₃; 2.2 to1.05, several m, residual aliphat. H; 1.70, s, 3H, acetyl; 1.23, d, 3H,Cβ-H Ala; 1.20, m, CH₃ isoprop.; 0.8, m, 3H, Cδ-H Nle

EXAMPLE 6

[0122] 3-(4-Fluorophenyl)propionyl-D-Nal(1)¹-Ser⁴-N-Me-Tyr5-D-Lys(Atz)⁶-Leu⁷-Arg⁸-Pro⁹-D-Ala¹⁰-NH₂

[0123] The synthesis was carried out according to a solid-phase flowdiagram (Peptide Synthesis Protocol, p. 11) with DIC/HOBt coupling,starting from 9.2 g of MBHA resin (loading density 1.08 mmol/g). AfterHF cleavage from the polymeric support, 5.8 g of crude peptide wereobtained, which were purified by standard processes of preparative HPLC.After subsequent freeze-drying, 2.0 g of HPLC-uniform unsubstitutedoctapeptide were obtained, of which 0.4 mmol was reacted with 0.5 mmolof 3-amino-1,2,4-trizole-5-carboxylic acid in the presence of PyBOP as acoupling reagent to give 790 mg of crude product of the desiredcompound. After fresh HPLC purification, 200 mg of target compound ofthe empirical formula C₆₄H₈₆N₁₇O₁₂F having correct FAB-MS: 1304.6 (M+H⁺)(calc: 1303.6) were obtained.

[0124]¹H-NMR (500 MHz, D₂O/DMSO-d₆, δ in ppm):

[0125] 8.14, m, 1H, 7.90, m, 1H, 7.80, m, 1H, 7.50, m, 2H, 7.35, m, 2H,7.0, m, 6H, 7.63, m, 2H, aromat. H; 5.0, m, 1H, 4.83, m, 2H, 4.41, m,1H, 4.30-4.05, several m, 4H, Cα-H; 3.66 to 2.25, several m, aliphat.and aromat. side-chain H; 2.95, s, and 2.75, s, N-Me; 2.05 to 1.1,several m, residual aliphat. H; 1.20, d, Cβ-H Ala; 0.75, m, 6H, Cδ-H Leu

EXAMPLE 7

[0126] Ac-D-Nal(2)¹-D-Cpa²-D-Pal(3)³-Ser⁴-N-Me-Tyr⁵-D-Lys(B)⁶-Nle⁷-Arg⁸-Pro⁹-Sar¹⁰-NH₂

[0127] The synthesis of the decapeptide was carried out on a polymericsupport with a loading density of 0.55 mmol/g (aminomethyl-substitutedresin, Fmoc protection, Type D-1675, Bachem). Lysine was coupled asFmoc-D-Lys(Boc)-OH, and the Fmoc protective groups were removed using20% piperidine/DMF. After simultaneous removal of all side-chainprotective groups and detachment from the polymeric support, theisolated crude peptide was purified by means of preparative HPLC. Afterfreeze-drying, 98.5% pure decapeptide was obtained.

[0128] The substitution on the ε nitrogen of D-lysine with4-(4-aminophenol)amino-1,4-dioxobutyric acid was carried out using PyBopin DMF with addition of DIPEA. The purification of the isolated crudepeptide was carried out by means of preparative HPLC. The subsequentfreeze-drying afforded about 99% pure product (trifluoroacetate) of theempirical formula C82H106ClN19O15 having correct FAB-MS of 1632 (M+H)(calc: 1631.78096)

EXAMPLE 8

[0129]Ac-D-Nal(2)¹-D-Cpa²-D-Pal(3)³-Ser⁴-N-Me-Tyr⁵-D-Lys(B)⁶-Nle⁷-Arg⁸-Pro⁹-D-Ala¹⁰-NH₂

[0130] The synthesis of the decapeptide was carried out on a polymericsupport with a loading density of 0.55 mmol/g (aminomethyl-substitutedresin, Fmoc protection, Type D-1675, Bachem). Lysine was coupled asFmoc-D-Lys(Boc)-OH, and the Fmoc protective groups were removed using20% piperidine/DMF. After simultaneous removal of all side-chainprotective groups and detachment from the polymeric support, theisolated crude peptide with a purity of about 71% (HPLC) was reactedfurther without purification.

[0131] The side-chain substitution of D-lysine with4-(4-aminophenol)amino-1,4-dioxobutyric acid was carried out using PyBopin DMF with addition of DIPEA. The isolated crude peptide was purifiedby means of preparative HPLC. After subsequent freeze-drying, a 98.8%pure product (trifluoroacetate) of the empirical formula C₈₂H₁₀₆ClN₁₉O₁₅having correct FAB-MS of 1632 (M+H) (calc: 1631.78096) was obtained.

EXAMPLE 9

[0132]Ac-D-Nal(2)¹-D-Cpa²-D-Pal(3)³-Ser⁴-N-Me-Tyr⁵-D-Lys(B)⁶-Nle⁷-Lys(iPr)⁸-Pro⁹-Sar¹⁰-NH₂

[0133] The synthesis of the decapeptide was carried out on a polymericsupport with a loading density of 0.55 mmol/g (aminomethyl-substitutedresin, Fmoc protection, Type D-1675, Bachem). Lysine was coupled asFmoc-D-Lys(Boc)-OH, and the Fmoc protective groups were removed using20% piperidine/DMF. After simultaneous removal of all side-chainprotective groups and detachment from the polymeric support, theisolated crude peptide (concentration about 59%, HPLC) was purified bymeans of preparative HPLC. After freeze-drying, 95% pure decapeptide wasobtained.

[0134] The side-chain substitution of D-lysine with4-(4-aminophenol)amino-1,4-dioxobutyric acid was carried out using PyBopin DMF with addition of DIPEA. The isolated crude peptide was purifiedby means of preparative HPLC. After subsequent freeze-drying, a 96.6%pure product (trifluoroacetate) of the empirical formula C₈₅H₁₁₂ClN17O₁₅having correct FAB-MS of 1646 (M+H) (calc: 1645.8218) was obtained.

[0135] The compounds according to formula I according to the inventionwere investigated for their receptor binding. The process closelyfollowed the process described in Beckers et al., Eur. J. Biochem. 231,535-543 (1995). Cetrorelix obtained according to the synthesis disclosedabove was iodinated with [¹²⁵I] (Amersham; specific activity 80.5Bq/fmol) using the IodoGen reagent (Pierce). The reaction mixture waspurified by reverse-phase high-performance liquid chromatography,monoiodinated cetrorelix being obtained without unlabelled peptide. Ineach case, about 80% of the [¹²⁵I]-cetrorelix and the unlabelledcompound according to the invention were suitable for the specificreceptor association.

[0136] The compounds according to the invention can be tested for theirin-vitro action using the following Methods 1 and 2, the bindingaffinities in the binding assay being determined with [¹²⁵I]-Cetrorelix(Method 1) and the functional activities being determined withtriptorelin as an agonist stimulus (Method 2).

[0137] Method 1.

[0138] Receptor binding assay according to Beckers, T., Marheineke, K.,Reilander, H., Hilgard P. (1995) “Selection and characterization ofmammalian cell lines with stable overexpression of human pituitaryreceptors for gonadoliberin (GnRH)” Eur. J. Biochem. 0.231, 535-543.

[0139] For investigation of the receptor binding, cetrorelix wasiodinated using the IodoGen reagent (Pierce) with [I] (Amersham; 80.5Bq/fmol specific activity). The reaction mixture was purified byhigh-performance liquid chromatography with exchanged phases,monoiodinated cetrorelix being obtained without unlabelled peptide.About 80% of the [¹²⁵I] cetrorelix was capable of specific receptorassociation.

[0140] The receptor binding assay was carried out under physiologicalconditions as described (Beckers et al., 1995) using intact cells.Subconfluent cultures of stably transfected LTK⁻ cells, which expressthe human LHRH receptor, were separated off by incubation in NaCl/P_(i)(137 mM NaCl, 2.7 mM KCl, 8.1 mM Na₂HPO₄, 11.47 mM KH₂PO₄)/1 mM EDTA andcollected by centrifugation. The cell pellet was resuspended in bindingbuffer (DMEM without H₂CO₃, with 4.5 g/l of glucose, 10 mM Hepes pH 7.5,0.5% (mass/volume) BSA, 1 g/l bacitracin, 0.1 g/l SBTI, 0.1%(mass/volume) NaN₃). For displacement assays, 0.25×10⁶ cells/100 μl wereincubated with approximately 225 pM of the [¹²⁵I]-cetrorelix (specificactivity 5-10×10⁵ dpm/pmol) and various concentrations of unlabelledcompound according to the invention as competitor. The cell suspensionin 100 pl of binding medium was layered in 400 μl assay tubes over 200μl of 84% by volume silicone oil (Merck Type 550)/16% by volume paraffinoil. After incubation for 1 h at 37° C. with slow, continuous shaking,the cells were separated from the incubation medium by centrifugationfor 2 min at 9000 rpm (rotor type HTA13.8; Heraeus Sepatec,Osterode/Germany). The tips of the tubes which contained the cell pelletwere cut off. Cell pellet and supernatants were then analysed bycounting the γ radiation. The amount of non-specifically bound materialwas determined at a final concentration of 1 μM with inclusion ofunlabelled cetrorelix and was typically ≦10% of the total boundmaterial. The analysis of the binding data was carried out using theEBDA/ligand analysis programme (Biosoft V3.0).

[0141] Method 2.

[0142] Functional Assay for the Determination of the AntagonisticActivity

[0143] The assay was carried out, provided with some modifications, asdescribed in Beckers, T., Reilander, H., Hilgard, P. (1997)“Characterization of gonadotropin-releasing hormone analogs based on asensitive cellular luciferase reporter gene assay”, Analyt. Biochem.251, 17-23 (Beckers et al., 1997). 10,000 cells per well, which expressthe human LHRH receptor and a luciferase reporter gene, were culturedfor 24 h in microtitre plates using DMEM with additives and 1% (v:v)FCS_(i). The cells were then stimulated with 1 nM [D-Trp⁶] LHRH for 6 h.Antagonistic compounds according to the invention were added before thestimulation and the cells were lysed at the end for the quantificationof the cellular Luc activity. The calculation of the IC₅₀ values fromdose-effect curves was carried out by non-linear regression analysisusing the Hill model (Programme EDX 2.0 from C. Grunwald,Arzneimittelwerk Dresden).

[0144] The quantification of the Luc activity was carried out induplicate essentially as described (Promega Technical Bulletins#101/161) using the respective luciferase assay system (Promega E4030).Owing to addition of coenzyme A (CoA), an oxidation of luciferyl-CoAtakes place with advantageous kinetics. After the removal of the culturemedium from the microtitre plate, the cells were lysed by addition of100 μl of lysis buffer (25 mM tris-phosphate pH 7.8, 2 mMdithiothreitol, 2 mM 1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid(CDTA), 10% (v:v) glycerol, 1% (v:v) Triton X-100). After incubation atroom temperature for 15 min, 10 μl of cell lysate were transferred intoa white microtitre plate suitable for luminometric detection (Dynatech).The enzymatic reaction was initiated by addition of 50 μl of assaybuffer (20 mM tricine pH 7.8, 1.07 mM (MgCO₃)₄Mg(OH)₂, 2.67 mM MgSO₄,0.1 mM ethylenediaminetetraacetic acid (EDTA), 33.3 mM dithiothreitol,270 μM coenzyme A, 470 μM glow-worm (Photinus pyralis) luciferin, 530 μMrATPNa₂). After one minute, the luminescence was determined for a totaltime of one second with a signal half-life of five minutes using theEG&G Berthold MicroLumat LB 96 P.

[0145] In this way, the following in-vitro data were obtained, K_(D)being the binding affinities and IC₅₀ being the functional activity andpM being picomoles per litre: Compound K_(D) [pM] IC₅₀ [pM] cetrorelix170 (21)  198 (5) Example 1 n.d.  242 (3) (Acetate salt) Example 2 181(1)  684 (2) Example 3 154 (1)  492 (2) Example 6 n.d.  221 (2) Example7 n.d. 1300 (1) Example 8 n.d. 1400 (1) Example 9 n.d. 4700 (1)

[0146]

1 1 1 10 PRT Artificial Sequence Description of Artificial SequenceIllustrative peptide 1 Glu His Trp Ser Tyr Gly Leu Arg Pro Gly 1 5 10

1-18. (Canceled)
 19. A compound of the general formula IA-Xxx¹-Xxx²-Xxx³-Xxx⁴-Xxx⁵-Xxx⁶-Xxx⁷-Xxx⁸-Xxx⁹-Xxx¹⁰-NH₂  (I) in which:A is an acetyl or a 3-(4-fluorophenyl)propionyl group, Xxx¹ is D-Nal(1)or D-Nal(2), Xxx²-Xxx³ is D-Cpa-d-Pal(3) or a single bond, Xxx⁴ is ser,Xxx⁵ is n-me-tyr, Xxx⁶ is D-[ε-N′-(imidazolidin-2-on-4-yl)formyl]-Lys,D-(3-amino-1,2,4-triazole-3-carbonyl)-Lys; orD-[ε-N′-4-(4-amidino-phenyl)amino-1,4-dioxobutyl]-Lys, Xxx⁷ is Leu orNle, Xxx⁸ is Arg or Lys(iPr), Xxx⁹ is Pro, and Xxx¹⁰ is Ala, D-Ala, orSar, and their salts with pharmaceutically acceptable acids:
 20. Thecompound according to claim 19, wherein the salt is an acetate,trifluoroacetate, or embonate.
 21. Method of treating prostate cancer orbreast cancer comprising administering an effective amount of compoundof the general formula IA-Xxx¹-Xxx²-Xxx³-Xxx⁴-Xxx⁵-Xxx⁶-Xxx⁷-Xxx⁸-Xxx⁹-Xxx¹⁰-NH₂  (I) in which:A is an acetyl or a 3-(4-fluorophenyl)propionyl group, Xxx¹ is D-Nal(1)or D-Nal(2), Xxx²-Xxx³ is D-Cpa-D-Pal(3) or a single bond, Xxx⁴ is Ser,Xxx⁵ is N-Me-Tyr, Xxx⁶ is D-[ε-N′-(imidazolidin-2-on-4-yl)formyl]-Lys,D-(3-amino-1,2,4-triazole-3-carbonyl)-Lys; orD-[ε-N′-4-(4-amidino-phenyl)amino-1,4-dioxobutyl]-Lys, Xxx⁷ is Leu orNle, Xxx⁸ is Arg or Lys(iPr), Xxx⁹ is Pro, and Xxx¹⁰ is Ala, D-Ala, orSar, and their salts with pharmaceutically acceptable acids to anindividual in need thereof.
 22. Method of treating benign prostatehyperplasia or endometriosis comprising administering an effectiveamount of compound of the general formula IA-Xxx¹-Xxx²-Xxx³-Xxx⁴-Xxx⁵-Xxx⁶-Xxx⁷-Xxx⁸-Xxx⁹-Xxx¹⁰-NH₂  (I) in which:A is an acetyl or a 3-(4-fluorophenyl)propionyl group, Xxx¹ is D-Nal(1)or D-Nal(2), Xxx²-Xxx³ is D-Cpa-D-Pal(3) or a single bond, Xxx⁴ is Ser,Xxx⁵ is N-Me-Tyr, Xxx⁶ is D-[ε-N′-(imidazolidin-2-on-4-yl)formyl]-Lys,D-(3-amino-1,2,4-triazole-3-carbonyl)-Lys; orD-[ε-N′-4-(4-amidino-phenyl)amino-1,4-dioxobutyl]-Lys, Xxx⁷ is Leu orNle, Xxx⁸ is Arg or Lys(iPr), Xxx⁹ is Pro, and Xxx¹⁰ is Ala, D-Ala, orSar, and their salts with pharmaceutically acceptable acids to anindividual in need thereof.